Integrins

Question 1

What are the components of the ECM?

Answer 1

• Cells (macrophages, fibroblasts, mast cells)
• Capillaries
• Proteins (collagen, fibronectin)
• Polysaccharides (glucosaminoglycans GAGs, heparin)
• Proteoglycans (decorin, CD44)

Question 2

What is the function of proteins in the ECM?

Answer 2

Tensile strength - collagen / elastin

Cell attachment - laminin / fibronectin

Question 3

Integrin function and family of proteins

Answer 3

Cell surface adhesion receptors that connect to actin / intermediate filaments by adaptor proteins.
18 alpha chain, 8 beta chains - 24 possible heterodimers.
All different functions

Question 4

Structure and function of fibronectin.

Answer 4

Two glycosylated chain linked by a S-S bond.
Roles in growth, development, wound healing and cancer.
Gene KO is lethal - highly conserved
Primary binding site = RGD motif, attaches to a5b1 integrin located on the 10F3 module.
This connection regulates cell attachment to the ECM.

Question 5

Explain the cell adherence and cell spreading assays and what they showed.

Answer 5

Adherence - Add cells, wash non-adherent, fluorescently label bound.
Any change to the RGD motif caused a loss of binding.

Spreading - fibronectin on plate, adheres and causes cells to spread. Count spread cells per unit area.
Only 9F3 and 10F3 showed spreading as well as whole fibronectin molecule.
Mutated 9F3 showed less spreading. Synergy Site = PHSRN important.
Engineered disulphide bond showed less spreading, needs flexibility about RGD loop.

Question 6

Multiple binding sites for cell spreading.

Answer 6

Fibronectin forms a mesh of fibrillar structures, enabling multiple RGD-integrin binding sites to be in close proximity to each other to cause spreading.

Question 7

Integrin structure

Answer 7

draw

Question 8

What are the three integrin conformational states

Answer 8

• Folded, masked binding sites. inactive
• Mid-state, crossed tails.
• Active, final state.

In the off state, the cytoplasmic tails are close together. They are further apart in the on-state. Distance regulates function.

Question 9

Two levels of control for integrin interaction strength.

Answer 9

• affinity regulation (interaction strength)

- valency (number of binding sites)

Question 10

Affinity regulation in aIIbB3

Answer 10

There are 3x MIDAS sites in integrin I-domains. Carboxyl groups provide a partial-charge to facilitate metal ion binding.
I-domains of integrins interact with RGD motif on ligand.
Causes a rearrangement in the MIDAS sites, increases affinity for the ligand and liberates the M335 at the top of a7 helix, causing it to move 7A up/down.
This causes a conformational change in the hybrid domain (bell-rope mechanism of activation), integrin tails move apart and integrin is active for outside-in signalling.

Question 11

What is the function of aIIbB3?

Answer 11

aIIbB3 - platelet integrin
Binds to fibrinogen / Von Willebrand factor to cause platelet aggregation.
Cytoskeletal engagement stabilises high affinity state.

Question 12

Regulation of valence

Answer 12

Redistribution (ligand induced, diffusion facilitated)
Microclustering (same as above)
Membrane Reshaping

All valency regulations increases the number on integrin contacts and therefore strengthens the integrin interaction to the ECM.

Question 13

Describe the cytosolic face of integrin receptors and example in aIIbB3.

Answer 13

Integrin tails are short and unstructured. They have multiple protein binding motifs.
aIIbB3 must be off in resting platelets - The aIIb is a negative regulator of the B3.
No aIIb tail gives a constitutively active integrin.

Question 14

What are proteins involved in focal adhesions?

Answer 14

Actin cytoskeleton
Structural (talin)
Signalling (FAK)
Adaptors (paxillin)

Question 15

What is the structure of talin and how is it activated?

Answer 15

Enables actin cross-linking
FERM - I/LWEQ domain structure.
FERM domain binds to the B-tail of the integrin and I’LWEQ bind the actin cytoskeleton.
Talin is kept in an inactive state - activated through PIs (reversible) or calpain cleavage (irreversible).

Question 16

Explain how talin causes inside-out signalling

Answer 16

Activation of talin from inside the cell causes the FERM domain to overcome the a-B interaction of the integrin tails to bind and activate the integrins, by separating tails.

Question 17

How can FRET be used to measure integrin tail separation.

Answer 17

One integrin tail (blue) will absorb light, while the other (yellow) will not.
If the integrin tails are close, when blue emits light, the energy profile will overlap with yellow, causing it to also emit light at different nm.
When the tails are separated, the profiles will not overlap and yellow will not emit light.

Question 18

How can talin be activated through mechanical stress and how can this be measured?

Answer 18

Under mechanical stress, talin undergoes the conformational change to expose the buried sequences so it can bind to focal adhesion proteins.
This can be measured using a cantilever, magnetic tweezers or GFP-labelled vinculin.

Question 19

Structure and function of focal adhesion kinase (FAK)

Answer 19

Only active in focal adhesions.
FERM - TyrK - FAT domain structure
TyrK can interact with PI3K via SH2 and FAT can interact with Grb2 and paxillin.
Usually in an auto-inhibited form - FERM blocks the kinase domain. Activated form - FERM and FAT dissociate from the active kinase.
B-integrins can activate FAK - downstream signalling for integrin clustering.